Proteases are among the most technically important enzymes of all. Their use in washing and cleaning agents has been established longer than that of any other enzymes, and they are contained in virtually all modern, high-performance washing and cleaning agents. They bring about the breakdown of protein-based stains on the item to be cleaned. Within this group, subtilisin-like proteases (subtilases, subtilopeptidases, EC 3.4.21.62), which because of the catalytically active amino acids are classed as serine proteases, are of particular importance. They act as non-specific endopeptidases and hydrolyze any acid amide bonds within peptides or proteins. Their pH optimum is usually in the distinctly alkaline range. A review of this family can be found for example in the article “Subtilases: Subtilisin-like Proteases” by R. Siezen, page 75-95 in “Subtilisin enzymes”, edited by R. Bott and C. Betzel, New York, 1996. Subtilisases are naturally formed by microorganisms. Particularly worthy of mention as the most important group within the subtilases are the subtilisins that are formed and secreted by Bacillus species.
Examples of the subtilisin-like proteases that are preferably used in washing and cleaning agents are the subtilisins BPN′ and Carlsberg, the protease PB92, the subtilisins 147 and 309, the protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY and the enzymes thermitase, proteinase K and the proteases TW3 and TW7, which can be assigned to the subtilases but are no longer subtilisins in the narrower sense, as well as variants of the specified proteases which have a modified amino acid sequence as compared with the starting protease. Proteases are modified by methods known from the prior art, either selectively or randomly, and thus optimized for use in washing and cleaning agents, for example. Such methods include point mutagenesis, deletion or insertion mutagenesis or fusion with other proteins or protein components. Correspondingly optimized variants are thus known for most proteases that are known from the prior art.
The international patent application WO 03/054185 discloses an alkaline protease from Bacillus gibsonii (DSM 14391), including the use thereof in washing or cleaning agents. In accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms of 28 Apr. 1977, this strain was deposited with the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, InhoffenstraBe 7B, 38124 Braunschweig, Germany on 1 Mar. 2001 under the ID number 01-192 and accession number DSM 14391. In contrast to the aforementioned proteases, this protease has considerable differences in the amino acid sequence, such that an identity comparison of the amino acid sequences results in identity values of below 80%. In the case of the alkaline protease from Bacillus gibsonii (DSM 14391), only a few protease variants optimized for use in washing and cleaning agents have been known in the prior art thus far.
The object of the present invention is to further develop a protease of the alkaline protease from Bacillus gibsonii (DSM 14391) type or a sufficiently similar protease (based on the sequence identity) and to obtain protease variants which are suitable and advantageously improved for use in washing or cleaning agents.
The invention provides a protease encompassing an amino acid sequence, which is at least 70% identical to the amino acid sequence specified in SEQ ID NO. 1 over the entire length thereof and which, in the listing according to SEQ ID NO. 1, has the amino acid substitution I21V in combination with at least one further amino acid substitution, the further amino acid substitution being selected from the group consisting of Q12L, M122L, N177V, A222S, V228I and T247N.
The invention also provides a method for producing a protease, encompassing the introduction of an amino acid substitution I21V in combination with at least one further amino acid substitution, the further amino acid substitution being selected from the group consisting of Q12L, M122L, N177V, A222S, V228I and T247N, in the listing according to SEQ ID NO. 1, into a starting protease, which over the entire length thereof is at least 70% identical to the amino acid sequence specified in SEQ ID NO. 1.
A protease within the meaning of the present patent application therefore encompasses both the protease as such and also a protease produced by a method according to the invention. Therefore all references to protease relate both to the protease as a substance and to the corresponding methods, in particular production methods for the protease.
Further subject matters of the invention relate to the proteases according to the invention and to methods for producing proteases according to the invention, to nucleic acids that code for these proteases, to non-human host cells containing proteases or nucleic acids according to the invention, and to agents encompassing proteases according to the invention, in particular washing and cleaning agents, to washing and cleaning methods, and to uses defined by means of proteases according to the invention.
Furthermore, other desirable features and characteristics of the present invention will become apparent from the subsequent detailed description of the invention and the appended claims, taken in conjunction with the accompanying drawings and this background of the invention.